Recombinant Subunit Rotavirus Trivalent Vaccine Candidate: Physicochemical Comparisons and Stability Evaluations of Three Protein Antigens.

Sanjeev Agarwal,Neha Sahni,David B. Volkin,Sangeeta B. Joshi,Ronald T. Toth,Stanley Cryz,Robert Sitrin,George A. Robertson,John M. Hickey

doi:10.1016/j.xphs.2019.08.002

Sanjeev Agarwal, Neha Sahni + Show 7 more

Open Access

https://doi.org/10.1016/j.xphs.2019.08.002

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Abstract

Although live attenuated Rotavirus (RV) vaccines are available globally to provide protection against enteric RV disease, efficacy is substantially lower in low- to middle-income settings leading to interest in alternative vaccines. One promising candidate is a trivalent nonreplicating RV vaccine, comprising 3 truncated RV VP8 subunit proteins fused to the P2 CD4+ epitope from tetanus toxin (P2-VP8-P[4/6/8]). A wide variety of analytical techniques were used to compare the physicochemical properties of these 3 recombinant fusion proteins. Various environmental stresses were used to evaluate antigen stability and elucidate degradation pathways. P2-VP8-P[4] and P2-VP8-P[6] displayed similar physical stability profiles as function of pH and temperature while P2-VP8-P[8] was relatively more stable. Forced degradation studies revealed similar chemical stability profiles with Met1 most susceptible to oxidation, the single Cys residue (at position 173/172) forming intermolecular disulfide bonds (P2-VP8-P[6] was most susceptible), and Asn7 undergoing the highest levels of deamidation. These results are visualized in a structural model of the nonreplicating RV antigens. The establishment of key structural attributes of each antigen, along with corresponding stability-indicating methods, have been applied to vaccine formulation development efforts (see companion paper), and will be utilized in future analytical comparability assessments.

Highlights

Rotavirus (RV) is a leading cause of acute diarrhea and gastroenteritis among infants and young children across the world with approximately 215,000 children under 5 years of age dying from RV infection each year.1 Most deaths occur in developing countries
As confirmed by LC-mass spectrometry (MS) peptide mapping results described below, an additional Met residue is present on the N-terminus of these antigens, as would be expected for a foreign protein expressed in E. coli
The potential impact of physicochemical structural alterations on immunogenicity is unknown at this time, developing such stability-indicating analytical tools and applying the structural knowledge gained in this work will be useful to (1) set critical manufacturing process parameters to ensure consistency, (2) monitor key structural attributes during comparability assessments, and (3) develop stable formulations for the bulk drug substance and adjuvanted final drug product

Summary

Introduction

Rotavirus (RV) is a leading cause of acute diarrhea and gastroenteritis among infants and young children across the world with approximately 215,000 children under 5 years of age dying from RV infection each year. Most deaths occur in developing countries. Rotavirus (RV) is a leading cause of acute diarrhea and gastroenteritis among infants and young children across the world with approximately 215,000 children under 5 years of age dying from RV infection each year.. Millions of children worldwide require home care, ER. Current address for Dr Agarwal: Amgen, Thousand Oaks, California 91320. Current addresses for Drs Sahni and Toth: GSK Vaccines, Rockville, Maryland 20850. Current address for Dr Robertson: Cambra Consulting, Woodbridge, Virginia 22039.

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